Mechanistic studies of transcription initiation by T7 RNA polymerase

Initiation of transcription is a central step in cellular regulation. In order to understand better the fundamental machanisms in this complex process, kinetic and structural studies have been carried out on a variety of modified promoter constructs using simple model RNA polymerase from bacteriophage T7. Investigation of the template strand elements in the initiation domain by introduction of non-nucleosidic spacers demonstrates that most of the apparent protein-DNA contacts in the crystal structure are not essential for initiation. In this respect, the part of the template strand from −4 to −2 serves as a “tether”, holding the templating bases near the active site. In cases where templating bases are not connected directly, the non-template strand duplexed downstream of the active site can serve a similar role. Some of the specificity in the positioning of the templating bases appears to be derived from the protein contact with the template strand base at position −1. Precise positioning of the templating bases near the active site appears to involve strong non-specific interactions of phosphodiester groups between positions −1 and +1, and +2 and +3. T7 RNA polymerase demonstrates strong preference for the initiating substrate nucleotide GTP. Experiments with the nucleotide analog 7–deaza–GTP demonstrate that this preference is due to a protein interaction with the non-Watson Crick face of the base involving the nitrogen-7 and oxygen-6 of guanine. In the presence of GTP as the sole substrate, on the template encoding GGGXX…,T7 RNA polymerase can engage in slippage transcription, making a ladder of RNA products ranging from 2 to about 14 nt in length. Termination of the ladder at 13–14 nt appears to involve a cooperative formation of structure in the nascent RNA, leading to the disruption of the transcribing complex. Slippage transcription from promoters encoding long runs of G provides evidence that the RNA:DNA heteroduplex initially can reach a length of nine nucleotides. The kinetics of synthesis of dinucleotide is characterized in a model system using numerical integration of mechanism-based rate equations. The analyses show that product rebinding cannot be ignored and can come to compete with the regular RNA production within a single enzyme turnover. Finally, the product release rate shows substrate dependence, suggesting that binding of an incoming nucleoside triphosphate can facilitate release of the bound RNA, most likely via direct competition at the active site